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Original Paper
Cellular Physiology
and Biochemistr
Biochemistryy
Cell Physiol Biochem 2003;13:173-180
Accepted: November 27, 2002
The Calpain-Calpastatin System and the Calcium
Paradox in the Isolated Perfused Pigeon Heart
Catherine Gaitanaki, Panagiota Papazafiri and Isidoros Beis*
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens,
Panepistimioupolis, Athens
Key Words
Calpain • Calcium paradox •Calpastatin •Pigeon heart
• Avian heart •Protection
Abstract
To examine whether the calpain-calpastatin system is
activated during the calcium paradox in the isolated
perfused pigeon heart, we separated the protease from
its inhibitor calpastatin and studied its kinetic
properties. The protease exhibits kinetic properties
similar to those of mammalian m-calpains. Ca 2+
requirements for half and maximum activities are 220
µM and 2 mM, respectively. In the absence of Ca2+
the protease is strongly activated by Mn2+ or Sr2+. In
the presence of Ca 2+ , Mn 2+ and Sr 2+ exhibit a
synergistic effect; Mg 2+ and Ba 2+ have no effect,
whereas Co2+, Ni2+ and Cd2+ completely inhibit its
activation. Furthermore, we measured the activity of
calpain and calpastatin under either conditions
inducing a calcium paradox, or protecting the heart
against this phenomenon. Although the calpain/
calpastatin ratio is lowered during Ca2+ depletion,
during Ca2+ repletion it is markedly inverted. Calpain
activation during reperfusion is inhibited by the
presence of 200 µM Mn2+ or Ba2+, in the Ca2+-free
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medium. Gel filtration of calpastatin, isolated from
either untreated hearts or during Ca2+ depletion,
produces two main peaks of ~150 and 40 kDa of
molecular mass, respectively, whereas calpastatin
isolated during the 2nd min of reperfusion appears to
be shifted to the 150 kDa form. All the above data
suggest that this system may be involved in the
induction of the calcium paradox in pigeon heart.
Copyright © 2003 S. Karger AG, Basel
Introduction
Calpain (Ca 2+ activated neutral protease), the
cysteine protease that absolutely require calcium for
catalytic activity, and its endogenous inhibitor calpastatin,
have been implicated in many physiological and
pathological conditions of increased protein degradation
associated with increased intracellular Ca2+ concentration
[1-5]. Altered calpain activities have also been measured
in myocardial injury, due to hereditary cardiomyopathy
[6] or ischaemia and reperfusion [7]. Upon
autoproteolytic activation, the protease can selectively
cleave a subset of cellular proteins including membrane
Dr. I. Beis
Dept. of Animal and Human Physiology, School of Biology
Faculty of Sciences, University of Athens
Panepistimioupolis, Athens 157 84 (Greece)
Tel. +30 10 7274 244, Fax +30 10 7274 635, E-Mail [email protected]
173
receptors, cytoskeletal proteins, and transciption factors.
Calpain is therefore well suited for executing messages
conveyed by shifting intracellular calcium concentrations
[4-5].
On the other hand, the Ca2+ paradox, namely the
unexpected heart necrosis during Ca 2+ repletion,
following a short period of Ca2+ deprivation [8], is caused
by a massive Ca2+ influx [9-10]. The Ca2+ paradox has
been experimentally induced in mammalian [8],
amphibian [11] and avian [12] myocardium and although
the exact mechanisms are uncertain, some major points
have been noted: First, a Ca2+ depletion results in an
increase of membrane permeability for Ca2+ [13-15].
Second, the Ca2+ influx during reperfusion leads to the
irreversible myocardial contraction (formation of
contraction bands), which in turn, is succeeded by
sarcolemmal ruptures, Ca2+ overload and eventually, cell
death [16-20].
Calpain activation, resulting from the massive Ca2+
influx, could be accounted for at least some of these
observations. First, Ca2+ overload has been correlated
with Z lines dissolution and/or myofibrillar disruption
[21-22] and calpain is localised to the Z lines of
myofibrils [23]. Second, contraction bands have also been
observed in post-ischaemic reperfusion of myocardium
[24] and these conditions are known to alter calpain
activity [7]. The common basis of these two phenomena
(the calcium and the oxygen paradox) has already been
underlined [25-26].
In our previous studies we had described the
characterisation of the calcium paradox [12], as well as
the protective effects of various divalent cations such as
manganese, barium, nickel, and cobalt against this
phenomenon in the isolated perfused pigeon heart
(unpublished data). The results of those studies clearly
showed that despite the fundamental structural and
functional differences between mammalian and avian
heart, the characteristics of this phenomenon induced
upon Ca2+ repletion following a 40 min Ca2+ depletion,
at normal body temperature (42 °C), are quite similar.
In the present study we examined the regulation of
the calpain-calpastatin system under conditions inducing
a calcium paradox as well as under conditions protecting
the pigeon heart against this phenomenon and provide
evidence for a potential role of this system in the calcium
paradox.
174
Cell Physiol Biochem 2003;13:173-180
Material and Methods
Animals
Isolated hearts of the pigeon Columba livia were used.
Domestic animals were obtained from a commercial dealer and
kept in the laboratory with free access to water and food. All
animals received humane care in accordance to the Guidelines
for the Care and Use of Laboratory Animals published by the
Greek government (160/1991) based on EC regulations (86/609).
Chemicals
The following materials were purchased from the sources
indicated: DEAE-cellulose DE-52 (Serva, Heidelberg, Germany);
Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden);
casein, Hammarsten grade (Merck, Darmstadt, Germany); E-64,
leupeptin, antipain, N-ethylmaleimide (NEM), phenyl methyl
sulfoxide (PMSF) (Sigma Chemical Co, St. Louis, U.S.A.). All
other chemicals used were of analytical grade and purchased from
Sigma Chemical Co.
Perfusion procedure
Pigeons (Columba livia) weighing 300-350 g were
anaesthetised with 20-25 mg sodium pentobarbital and received
heparin (400 IU) intravenously. The hearts were excised and
mounted onto the aortic cannula of a conventional Langendorff
perfusion system. All perfusions were carried out at a constant
perfusion pressure of 90 cm H2O and a flow of 15-18 ml/min.
The normal perfusion medium was a Krebs-Henseleit’s (KH)
bicarbonate buffer which consisted of (in mM): 118 NaCl, 2.96
KCl, 1.2 MgSO4, 1.2 KH2PO4, 2 CaCl2, 25 NaHCO3, 10 glucose
and 1 sodium pyruvate. The pH of the oxygenated KH buffer was
adjusted at 42°C to 7.35-7.40. All KH buffers were equilibrated
with 95% O2-5% CO2. In the calcium-free medium calcium was
omitted and EGTA was added at a final concentration of 10 µM
to ensure removal of any contaminant calcium. In different sets
of experiments 200 µM of BaCl2, or MnCl2, along with 10 µM
EGTA were added in the calcium free KH buffer. In all
experiments an equilibration period of 15 min was allowed during
which the hearts were perfused with the normal KH buffer. This
was followed by a 40 min period of calcium depletion in the
presence or absence of added divalent cations and finally by the
reperfusion with standard KH buffer for increasing time intervals.
At the end of perfusion, the hearts were rapidly frozen at -170 °C
and the ventricles were removed and stored at -80 °C.
Partial purification of calpain
All purification procedures were carried out at 2-4 °C. The
frozen ventricles (4-5 g of weight) were ground to powder under
liquid nitrogen and homogenized in 5 volumes of Tris-EGTA (TE)
buffer, which contained (in mM): 20 Tris-HCl, pH 7.4, 1 EGTA,
5 NaN 3 and 10 2-mercaptoethanol. The homogenate was
centrifuged at 11,000 x g for 30 min and the supernatant was
precipitated by a 30% ammonium sulfate saturation (16.4 g/100
ml), stirred for 30 min and centrifuged at 11,000 x g for 20 min.
The supernatant was then brought to a 70% ammonium sulfate
saturation (24.9 g/100 ml), stirred for 60 min and centrifuged at
11,000 x g for 20 min. The precipitate was suspended in TE buffer
Gaitanaki/Papazafiri/Beis
Fig. 1. Representative elution
profile of calpain and calpastatin
from the DEAE cellulose DE-52
column as determined by monitoring
for protein and calpastatin and
calpain activities. The arrows
indicate the application time of the
different, NaCl containing, TE
buffer.
and salt excess was removed by overnight dialysis against an
excess volume of the same buffer. The suspension was then loaded
onto a DEAE cellulose DE 52 column (20 ml packed volume,
equilibrated with TE buffer). After being washed with 60 ml of
TE buffer, the column was eluted with 80 ml of TE buffer, which
contained 150 mM NaCl followed by 80 ml of TE buffer
containing 400 mM NaCl. The eluted fractions (5 ml) were
monitored for protein (A280) and calpain and calpastatin activities.
The calpain active fractions were pooled, dialysed overnight in
TE buffer and concentrated by ultrafiltration using an Amicon
membrane (PM30). This final enzyme preparation was stored at 20 °C in 50% (v/v) glycerol for at most 3 weeks, without any loss
of calpain activity during this period. Fractions of the final
preparation were dialysed in 20 mM imidazole-HCl buffer, pH
7.4, 50 µM EGTA, 10 mM 2-mercaptoethanol and 10% (v/v)
glycerol, a few hours before the assay of calpain activity was
performed.
Subcellular fractionation
Homogenates (1 g of tissue), prepared as described above,
were centrifuged at 11,000 x g for 30 min and the supernatants
were then centrifuged at 100,000 x g for 60 min. The particulate
fractions were suspended in TE buffer. The suspension of the
11,000 and 100,000 x g pellets and the 100,000 x g supernatants
were subjected to DEAE cellulose columns (2 ml of packed
volume, equilibrated with TE buffer). Fractions (1 ml) were
collected as described above and monitored for calpain and
calpastatin activities.
Protein determination
Protein was determined by the method of Lowry et al. [27],
using bovine serum albumin as the standard.
Calpain-Calpastatin System and Calcium Paradox
Assay of calpain and calpastatin activity
Calpain activity was measured as a release of peptides from
alkali denatured casein, as described by Ishiura et al. [28], with
slight modifications [29]. The standard assay mixture (0.5 ml)
contained 50 mM imidazole-HCl buffer, pH 7.4, 10 mM 2mercaptoethanol, 5 mg/ml alkali denatured casein, 5 mM CaCl2
and 20-100 µl of each sample. Standard assay mixtures without
the sample or with 5 mM EGTA instead of CaCl2 were used as
blanks. The mixtures were incubated at 30 °C for 60 min and the
reactions were terminated by the addition of 0.5 ml of 10% (w/v)
trichloroacetic acid (TCA) solution. The samples were allowed
to stand on ice for 30 min and then centrifuged at 11,000 x g for
8 min. The proteolytic products were measured at 750 nm by the
method of Ross and Schatz [30]. For this, 0.2 ml of the supernatant
was mixed with the reagents and diluted to a final volume of 3.25
ml. One unit of calpain activity was defined as an increase in A750
of 1.0 per ml of sample per hour. The determination of calpastatin
activity was performed with a fixed amount of calpain and one
unit of the inhibitor was defined as a decrease in A750 of 1.0 under
standard assay conditions.
Gel filtration
Calpastatin molecular weight was assessed by gel filtration,
performed as described by Whitaker [31], with slight
modifications. Samples (2 ml final volume containing 0.5 M
sucrose) from the calpastatin active fractions, eluted from the
DEAE cellulose column, were applied to a Sephadex G-200
column (0.70 x 32.5 cm) and the eluted (with TE buffer, at 4 °C
and ionic strength of 0.200) fractions (2 ml) were monitored for
calpastatin activity. The void volume was determined by using
Blue Dextran 2000, mixed with 0.5 M sucrose and calpastatin
Cell Physiol Biochem 2003;13:173-180
175
Fig. 2. Effect of Ca2+
concentration on calpain activity and synergistic effect of Mn2+.
The activity measured
at 2 mM Ca 2+ was
considered as 100 %.
SEM are less than
0.02% of mean values.
Results
Table 1. Activation, inhibition of calpain activity, as well as
synergistic effect of various divalent cations. The activity
measured at 5 mM Ca2+ was considered as 100 %. Values are
the mean±SEM of 5 determinations
molecular weight was estimated by using gel filtration protein
markers: apoferritin (443 kDa), alcohol dehydrogenase (150 kDa)
and carbonic anhydrase (29 kDa), also mixed with 0.5 M sucrose.
Chromatographic separation of calpain from
calpastatin
A) Separation of calpain from calpastatin. Calpain
activity, measured in myocardium extracts without the
DEAE cellulose chromatographic procedure, was not
detectable, due to the presence of calpastatin. The
determination of calpain and calpastatin activities in the
11,000 x g supernatant eluted from the DEAE cellulose
column, showed that calpastatin is eluted by 0-150 mM
NaCl, in TE buffer, and completely separated from
calpain, which is eluted by 150-400 mM NaCl, in the
same buffer (Fig. 1).
B) Fractional localisation of calpain. The activity
found at the 11,000 x g supernatant was the same with
the activity measured at the 100,000 x g supernatant
following the same chromatographic procedure, while
there was no, detectable with the employed procedure,
calpain or calpastatin activity at the 11,000 and 100,000
x g particulate fractions (data not shown).
Data analysis
The results are presented as mean±SE of 4-5 independent
experiments. The statistical significance was determined by using
the Student’s unpaired t-test at P<0.05 level of confidence.
Properties of partially purified calpain
A) Effect of various divalent cations. Incubation of
calpain with increased Ca2+ concentrations showed that
maximum and half-maximum calpain activities are
attained at 2 mM and 220 µM Ca2+ respectively (Fig. 2).
No activity is detected at 50 µM Ca2+ indicating that the
176
Gaitanaki/Papazafiri/Beis
Cell Physiol Biochem 2003;13:173-180
Table 2. Required concentration of the
inhibitors for 50% inhibition of calpain
activity. Values are the mean ±SEM of four
determinations.
Table 3. Effect of Ca2+ depletion and reperfusion on
calpain and calpastatin total activities measured in the
DEAE cellulose fractions. Values are mean± SEM of
four different determinations. *, P<0.05; **, P<0.001
versus control. (1) 200 µM of each cation were added
during Ca2+ depletion.
enzyme is a millimolar isoform (m-calpain). As shown
in Table 1, at 5 mM, only Mn2+ and Sr2+, activate calpain,
substituting for Ca 2+, which activates calpain to a
maximum extent. Furthermore, in the presence of 1 mM
Ca2+, Mn2+ and Sr2+, show a synergistic effect (Table 1).
Among the other cations that do not activate calpain,
Mg2+ and Ba2+ do not inhibit calpain activation by Ca2+
whereas Co2+, Ni2+ and Cd2+ completely inhibit calpain
activation, showing an effect of inhibition by this group
of cations.
In the presence of increased Ca2+ concentrations
(Fig. 2), Mn2+ (400 µM) causes a decrease in Ca2+
requirement, from 220 µM to 160 µM and from 2.0 to
0.5 mM (for half-maximum and maximum activity,
respectively). Neither cation showed any synergistic
effect at 200 or 300 µM (data not shown).
B) Effect of various protease inhibitors. In order to
study the effect of various protease inhibitors on calpain
activity, standard assay conditions were used. Inhibition
grade was expressed as a % decrease in calpain activity,
considering the activity measured in the absence of the
inhibitors as 100%. Thiol-protease inhibitors, such as
E-64, leupeptin and antipain, strongly inhibit calpain
activity (Table 2), at a concentration lower than 10 µM.
The alkylating reagent, N-ethylmaleimide (NEM), also
inhibits calpain activity but at a much higher
concentration (1 mM). PMSF even at 3 mM, has no
significant inhibitory effect (Table 2), indicating that this
enzyme is a member of the thiol-protease group.
C) Effect of the Ca2+ paradox.. The effect of the
2+
Ca paradox on calpain activity was tested by perfusion
of the hearts for 40 min with a Ca2+-free medium, in the
presence or absence of 200 µM Mn 2+ or Ba2+, and
subsequent reperfusion with the Ca2+ containing KH
buffer. The hearts were frozen in specific intervals and
calpain activity was measured.
In Table 3 calpain and calpastatin total activities
measured at the DEAE cellulose fraction, eluted by 400
and 150 mM NaCl, respectively are shown. During Ca2+
depletion both enzyme and its endogenous inhibitor (to
a larger extent) activities linearly increase. The presence
of Mn2+ or Ba2+ during this period has no effect on the
observed calpain and calpastatin activation.
During calcium repletion followed a 40 min calcium
depletion however, calpain activity is continuously raised
to a maximum attained at the 2nd min of reperfusion, while
calpastatin activity is lowered at the first 30 sec of
reperfusion to be raised to a maximum also attained at
the 2 nd min (Table 3). Calpain activation during
reperfusion is inhibited by the presence of 200 µM Mn2+
Calpain-Calpastatin System and Calcium Paradox
Cell Physiol Biochem 2003;13:173-180
177
or Ba 2+, in the Ca2+-free medium, while calpastatin
activation is inhibited only by Mn2+.
The presence of Mn2+ or Ba2+, at 200 µM, during
2+
Ca depletion, powerfully protects the heart (recovery
of mechanical and maintenance of the electrical activity
of the heart) against the induction of the Ca2+ paradox
(Gaitanaki et al., unpublished data).
Although calpain/calpastatin ratio is lowered during
Ca2+ depletion, during reperfusion it is markedly inverted,
with the maximum value attained at the first min of
reperfusion (Fig. 3).
D) Calpastatin molecular mass. In order to estimate
whether the alteration of calpastatin activity is mainly
due to diffusional loss and/or intracellular translocation,
or it is the result of affected equilibrium between two
different calpastatin forms, we assessed calpastatin
molecular mass during Ca2+ depletion and reperfusion.
Gel filtration of calpastatin, isolated from untreated
hearts, produces two main peaks of calpastatin activity
(Fig. 4A) at 150 and 40 kDa approximate molecular mass,
respectively. The recovery of calpastatin activity was in
every case over 98%. Calpastatin isolated at the end of
Ca2+ depletion shows an almost identical elution pattern
(Fig. 4B), while the ratio of the two different molecular
mass forms, of calpastatin isolated at the 2nd min of
reperfusion, appears to be shifted to the 150 kDa form
(Fig. 4C).
Fig. 3. The alteration of calpain/calpastatin ratio during Ca2+
depletion (A) and reperfusion (B). After a relative decrease during
the first period the ratio is inverted at the second period with
calpain exceeding calpastatin.
Fig. 4. Elution
pattern on gel filtration, of calpastatin isolated from
control (A), Ca 2+
depleted (B), and
reperfused (C)
hearts. The numbers under the peaks indicate the (%)
ratio of each calpastatin form.
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Cell Physiol Biochem 2003;13:173-180
Gaitanaki/Papazafiri/Beis
Discussion
The alteration of calpain and calpastatin activities,
demonstrated by this study, during Ca2+ depletion and
reperfusion, indicates that this Ca2+ activated neutral
protease system might be implicated in the Ca2+ paradox.
The millimolar form of calpain, isolated from the
pigeon myocardium by ion exchange chromatography
(Fig. 1), is activated by high Mn2+ and Ba2+ concentration
(Table 1), similarly to m-calpain from other sources [29,
32], but differently to others [28, 33]. The synergistic
effect of Mn2+ and Ca2+ (Fig. 2, Table 1) is also in
agreement with other reports [34-35].
Measurements of calpain and calpastatin activities
at the end of the Ca2+ depletion and during the Ca2+
repletion and comparison with those under physiological
conditions (Table 3), shows that moderately the enzyme
and mostly the inhibitor are activated after Ca2+ depletion.
This activation is probably indirect, due to a decreased
intracellular Ca2+ concentration, since the presence of
Ca2+ is considered to be necessary for enzyme and
inhibitor interaction [36-37]. Furthermore, a functionally
active fraction of the endogenous inhibitor, known to be
associated with membranes [38-39], could be released
by structural changes of sarcolemmal phospholipids and
carbohydrates, which are observed during Ca2+ depletion
[40-41]. In particular, the plasma membrane phospholipid
turnover has been shown to be stimulated in mammalian
myocardium during ischaemia or reperfusion [42]. It
seems therefore that an elevated phosphoinositol level
may also contribute to the activation of the calpaincalpastatin system in our experimental model.
Calpain activity continuously increases (Table 3)
during the first 2 minutes of reperfusion, probably due
to a massive Ca2+ influx. Mn2+ and Ba2+, at a concentration
(200 µM) that powerfully protects the heart against the
induction of a Ca2+ paradox and having no direct effect
on calpain activity, appear to inhibit calpain activation
during reperfusion, reducing, each with a different
possible mechanism, the Ca2+ influx. Although the
reduction of calpastatin activity at the first 30 sec of
reperfusion (Table 3) probably indicates diffusional loss
or association with membranes, the inhibitor is also
activated at the 2nd min of reperfusion, without, however
preventing the inversion of calpain/calpastatin ratio (Fig.
3). Mn2+ appears to inhibit calpastatin activation during
reperfusion, while Ba2+ does not (Table 3); the protective
action of each cation, against a Ca2+ paradox, is also
considered to be resulting of a different mechanism [4344]. Finally, the loss of calpain and calpastatin activities
at the third min of reperfusion (Table 3) possibly
coincides with irreversible damages of cardiac cells,
following the massive Ca2+ influx.
Although calpastatin appears to migrate
anomalously on gel filtration [45-46], two main peaks of
inhibitory activity are found (Fig. 4). The assessed
molecular masses of 150 and 40 kDa, are quite
comparable to the two most commonly reported values:
120 and 70 kDa [5]. It is uncertain whether multiple forms
of calpastatin exist in vivo or they are the product of
differential susceptibility to proteolysis during
purification procedure. Nevertheless, the altered ratio of
the two forms, at the 2nd min of reperfusion, could also
be explained by a possible association with membranes
of the smaller calpastatin molecules at the first 30 sec of
reperfusion.
In conclusion, the calpain system could possibly
provide a basis for the elucidation of the Ca2+ paradox
mechanism, either by degrading myofibrillar or
cytoskeletal proteins, indispensable for conservation of
contractile capability and sarcolemmal integrity or by
activating Ca2+ dependent enzymes that initiate an
irreversible, self destructive pathway. However, further
studies on this protease and inhibitory protein system,
with cell permeable, calpain specific inhibitors are
necessary for the clarification of this system’s
participation in the Ca2+ paradox induction.
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